负载抗生素铜掺杂羟基磷灰石微球的制备及抗菌成骨评价

Preparation of Antibiotic-Loaded Copper-Doped Hydroxyapatite Microspheres and Evaluation of Their Antibacterial and Osteogenic Effect

目的探讨负载万古霉素(vancomycin,Van)的铜(Cu)掺杂羟基磷灰石(hydroxyapatite,HA)微球制备方法,并于体外评价其抗菌及成骨作用。方法采用水热合成法制备摩尔掺杂比例1%、5%、10%、20%的Cu掺杂HA微球(CuHA),通过扫描电镜观察其微观形貌变化,X射线衍射仪(X-ray diffractometer,XRD)研究其相组成并分析样品的结晶度。选择摩尔掺杂比例为10%的Cu-HA,利用X射线能谱仪(energy dispersive X-ray spectroscopy,EDS)分析样品的元素成分信息后,以聚多巴胺(polydopamine,PDA)为介质涂覆制备Cu-HA-PDA,采用XRD和傅里叶红外光谱仪检测样品涂覆效果。在Cu-HA-PDA装载Van,制备成Cu-HA-PDA-Van。将均为10 mg/mL的HA、Cu-HA、HA-PDA、Cu-HA-PDA-Van分别加入α培养基中制备各组浸提液,检测浸提液主要成分,检查Van质量浓度。探究材料浸提液对成骨前体细胞的毒性作用及骨髓间充质干细胞的增殖、分化作用,并检测成骨相关基因骨钙素(osteocalcin,OCN)、Runt相关转录因子2(runt-related transcription factor 2,RUNX-2)、碱性磷酸酶(alkaline phosphatase,ALP)表达。备好灭菌的HA、Cu-HA、HA-PDA、Cu-HAPDA、Cu-HA-DPA-Van微球材料,采用菌落计数法评价材料对金黄色葡萄球菌的抗菌效果。结果成功合成各种CuHA,随着Cu比例的增加,其形貌从条带状逐渐变成均匀球状,其中摩尔掺杂比例为10%的Cu-HA显示良好的微球形状及表面花瓣状多孔微纳形貌。EDS及XRD分析表明材料主体结构依然为羟基磷灰石晶体且Cu已成功掺杂HA。傅里叶红外光谱仪显示PDA成功涂覆在材料表面。浸提液主要成分检测再次佐证Cu元素成功进入并替代部分HA中的Ca元素。10 mg/mL Cu-HA-PDA-Van浸提液中含Van 0.27 mg/mL。体外细胞实验和成骨相关基因检测表明,Cu-HA-Van具有良好的生物活性和促骨分化作用。Cu-HA-PDA-Van微球最小抑菌浓度(minimum inhibitory concentration,MIC)为16μg/mL,Cu-HA-Van微球相对于Cu-HA、HA-PDA和纯HA有显著和持久的抗菌效果。结论利用Cu元素调控HA微观形貌、成功涂覆PDA并装载Van制备的Cu-HA-PDA-Van微球具有良好的抗菌性能和生物活性。

Objective To explore the preparation method of copper(Cu)-doped hydroxyapatite(HA microspheres loaded with vancomycin(Van),and evaluate their antibacterial and osteogenic effects in vitro.Methods The Cu doped HA microspheres(Cu-HA)with molar doping ratios of 1%,5%,10%,and 20%were prepared by hydrothermal synthesis.The microscopic morphology changes were observe with scanning electron microscope.X-ray diffractometer(XRD)was used to study the phase composition and analyze the crystallinity of the sample.Cu-HA with a molar doping ratio of 10%was selected for analysis of the elemental composition of the sample with energy dispersive Xray spectroscopy(EDS),and was then coated with polydopamine(PDA)as the medium to prepare Cu-HA-PDA.XRD and Fourier infrared spectrometer were used to examine the coating effect of the sample.Van was load on Cu-HA-PDA to prepare Cu-HA-PDA-Van.HA,Cu-HA,HA-PDA,and Cu-HA-PDA-Van were added toαmedium at 10 mg/mL to prepare different groups of extract solutions.The main components of the extract solutions were examined,and the Van concentration was checked.We examined the toxic effect of material extract solutions on osteogenic precursor cells and the proliferation and differentiation of bone marrow mesenchymal stem cells,and checked the expression of osteocalcin(OCN),runt-related transcription factor 2(RUNX-2),and alkaline phosphatase(ALP),the osteogenic related genes.Sterilized HA,Cu-HA,HA-PDA,Cu-HA-PDA,Cu-HA-DPA-Van microsphere materials were prepared,and the colony counting method was used to evaluate the antibacterial effect of the materials for Staphylococcus aureus.Results Various types of Cu-doped HA(Cu-HA)were successfully synthesized.As the proportion of Cu increased,the morphology gradually changed from being strip or belt-shaped to a uniform spherical shape.Cu-HA of 10%molar doping ratio showed a clearly microspherical shape and a petal-like porous micro-nano morphology on the surface.EDS and XRD analyses showed that the main structure of the material was still made up of hydroxyapatite crystals and Cu was successfully doped with HA.The infrared spectrometer showed that the PDA was successfully coated on the surface of the material.Examination of the main components of the extract solution once again verified that the Cu element had successfully entered and replaced part of the Ca element in the HA.The 10 mg/mL Cu-HA-PDA-Van extract solution contained 0.27 mg/mL of Van.In vitro cell experiments and bone-formation-related gene testing showed that Cu-HAVan had good biological activity and promoted bone differentiation.The minimum inhibitory concentration(MIC)of Cu-HA-PDA-Van microspheres was 16μg/mL.Compared with Cu-HA,HA-PDA and pure HA,Cu-HA-Van microspheres had significant and long-lasting antibacterial effects.Conclusion Cu element was used to control the microscopic morphology of HA,and the Cu-HA-PDA-Van microspheres prepared by successfully coating of PDA and loading of Van had good antibacterial properties and biological activity.